Methods for modulating inflammasome activity and inflammation in the lung

ABSTRACT

The present invention provides compositions and methods for reducing inflammation in the lungs of a mammal that is afflicted by a condition that leads to inflammation in the lungs. The compositions and methods described herein include agents that inhibit inflammasome signaling in the mammal such as antibodies directed against inflammasome components used alone or in combination with extracellular vesicle uptake inhibitor(s).

This application claims priority from U.S. Provisional ApplicationSerial No. 62/440,180, filed Dec. 29, 2016, which is herein incorporatedby reference in its entirety for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under grant number4R42BS086274-02 awarded by the National Institute of NeurologicalDisorders and Stroke (NINDS). The U.S. government has certain rights inthe invention.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:UNMI_010_01WO_SeqList_ST25.txt, date recorded: Dec. 28, 2017, file size2 kilobytes).

FIELD

The invention relates generally to the fields of immunology andmedicine. More particularly, the invention relates to compositions andmethods for modulating ASC (Apoptosis-associated Speck-like proteincontaining a Caspase Activating Recruitment Domain (CARD)) activity andAbsent in Melanoma 2 (AIM2) inflammasome activity in the lungs of amammal as treatments for reducing inflammation in response to conditionsthat produce inflammation in the lungs.

BACKGROUND

Severe Traumatic Brain Injury (TBI) is a major public health concern andis a leading cause of mortality and morbidity throughout the world (3).In addition to direct injury to the brain, TBI may lead to complicationsin other organs, such as the lungs. Acute Lung Injury (ALI; 2) is acommon cardiopulmonary problem after trauma and is associated with ahospital mortality rate of up from 40% (4). TBI patients, in particular,are susceptible to develop ALI, with some studies reporting an incidenceas high as 30% (5). Recent studies have shown that systemic inflammatoryfactors may lead to pulmonary dysfunction and lung injury after TBI (6),but the precise molecular mechanism underlying TBI-induced lung injuryremain poorly defined.

A flood of secreted inflammatory mediators, including cytokines,chemokines, and damage-associated molecular patterns (DAMPs) released byinjured cells contribute to brain inflammation and affect distal organssuch as the lungs (5). One of the most widely studied DAMPs is the highmobility group box-1 (HMGB1), which can serve as an early mediator ofinflammation in various pathogenic states, including TBI (7). A morerecent study has shown that HMGB1 can be involved in the mechanism ofTBI-induced pulmonary dysfunction (8). HMGB1 release can be regulated bythe inflammasome (9), a multi-protein complex involved in the activationof caspase-1 and the processing of IL-1β and IL-18 after TBI (10).

A variety explanations have been put forth to explain pathomechanisms ofpulmonary complications after TBI, including increased vascularpermeability leading to capillary leakage and infiltration ofproteinaceous debris (11). Extracellular vesicles (EV) aremembrane-contained vesicles that play a role in cell-to-cellcommunication (12) and have been implicated to play a role in thedevelopment ALI in a LPS-induced murine model. Further, it has beenshown that EV can carry bioactive cytokines such as IL-1β andinflammasome proteins (13) (14), and may trigger an immune response andamplify inflammation via its cargo to neighboring and surrounding cells.However, it is unknown if EV-mediated inflammasome signaling cancontribute to the pathomechanism of TBI-induced ALI. Further, it is alsounknown whether the pathomechanisms of TBI-induced ALI are shared byother conditions that produce lung inflammation. In addition, there is ascarcity of Federal Drug Administration (FDA) approved drugs to treatlung inflammation.

Accordingly, there is an urgent need not only for elucidating thepathomechanisms of lung inflammation caused by TBI as well as otherconditions, but also the development of therapeutic compositions anduses thereof for treating and/or preventing lung inflammation.

SUMMARY

In one aspect, provided herein is a method of treating inflammation inlungs of a patient in need thereof, the method comprising: administeringto the patient a composition comprising an agent that inhibitsinflammasome signaling, whereby the inflammation in the lungs of thepatient is treated. In some cases, the inflammation in the lungs iscaused by a condition selected from a central nervous system (CNS)injury, a neurodegenerative disease, an autoimmune disease, asthma,chronic obstructive pulmonary disease, cystic fibrosis, interstitiallung disease and acute respiratory distress syndrome. In some cases, theCNS injury is selected from the group consisting of traumatic braininjury (TBI), stroke and spinal cord injury (SCI). In some cases, theneurodegenerative disease is selected from the group consisting ofamyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) andParkinson’s disease (PD). In some cases, the administration of thecomposition results in inhibition of inflammasome activation in lungcells of the patient. In some cases, the administration of thecomposition results in a reduction of caspase-1, nucleotide-bindingleucine-rich repeat pyrin domain containing protein 1 (NLRP1),nucleotide-binding leucine-rich repeat pyrin domain containing protein 2(NLRP2), nucleotide-binding leucine-rich repeat pyrin domain containingprotein 3 (NLRP3), NLR family CARD domain-containing protein 4 (NLRC4),caspase-11, X-linked inhibitor of apoptosis protein (XIAP), pannexin-1,Apoptosis-associated Spec-like protein containing a Caspase ActivatingRecruitment Domain (ASC), interleukin-18 (IL-18), high mobility groupbox 1 (HMGB1) or absent in melanoma 2 (AIM2) levels in lung cells of thepatient as compared to a control, wherein the control is an untreatedpatient. In some cases, the lung cells are Type II alveolar cells. Insome cases, the administration of the composition results in a reductionin acute lung injury (ALI) as compared to a control, wherein the controlis an untreated patient. In some cases, the reduction in ALI isevidenced by a reduction in neutrophil infiltration into alveolar and/orinterstitial space, reduced or absent alveolar septal thickening or acombination thereof. In some cases, the agent is an extracellularvesicle (EV) uptake inhibitor, an antibody that binds to an inflammasomecomponent or a combination thereof. In some cases, the EV uptakeinhibitor is a compound or an antibody, wherein the antibody is selectedfrom Table 1. In some cases, the agent is an EV uptake inhibitor incombination with an antibody that binds to an inflammasome component. Insome cases, the EV uptake inhibitor is a heparin. In some cases, theheparin is Enoxaparin. In some cases, the antibody that binds to aninflammasome component is an antibody that specifically binds to acomponent of a mammalian AIM2, NLRP1, NLRP2, NLRP3 or NLRC4inflammasome. In some cases, the inflammasome component is caspase-1,ASC or AIM2. In some cases, the inflammasome component is ASC. In somecases, the antibody binds to an N-terminal PYRIN-PAAD-DAPIN domain(PYD), C-terminal caspase-recruitment domain (CARD) domain or an epitopederived from the PYD or CARD domain of the ASC protein. In some cases,the antibody binds to a protein having at least 85% sequence identitywith an amino acid sequence selected from the group consisting of SEQ IDNO: 1 and SEQ ID NO: 2. In some cases, the antibody inhibits ASCactivity in the lungs of the patient. In some cases, the composition isformulated with a pharmaceutically acceptable carrier or diluent. Insome cases, the composition is administered intracerebroventricularly,intraperitoneally, intravenously or by inhalation.

In another aspect, provided herein is a method of treating inflammationin lungs of a patient that has been subjected to a central nervoussystem (CNS) injury, the method comprising: administering to the patienta composition comprising an agent that inhibits inflammasome signaling,whereby the inflammation in the lungs of the patient is treated. In somecases, the CNS injury is selected from the group consisting of traumaticbrain injury (TBI), stroke and spinal cord injury (SCI). In some cases,the administration of the composition results in inhibition ofinflammasome activation in lung cells of the patient. In some cases, theadministration of the composition results in a reduction of caspase-1,NLRP1, NLRP2, NLRP3, NLRC4, caspase-11, XIAP, pannexin-1,Apoptosis-associated Spec-like protein containing a Caspase ActivatingRecruitment Domain (ASC), interleukin-18 (IL-18), high mobility groupbox 1 (HMGB1) or absent in melanoma 2 (AIM2) levels in lung cells of thepatient as compared to a control, wherein the control is an untreatedpatient. In some cases, the lung cells are Type II alveolar cells. Insome cases, the administration of the composition results in a reductionin acute lung injury (ALI) as compared to a control, wherein the controlis an untreated patient. In some cases, the reduction in ALI isevidenced by a reduction in neutrophil infiltration into alveolar and/orinterstitial space, reduced or absent alveolar septal thickening or acombination thereof. In some cases, the agent is an extracellularvesicle (EV) uptake inhibitor, an antibody that binds to an inflammasomecomponent or a combination thereof. In some cases, the EV uptakeinhibitor is a compound or an antibody, wherein the antibody is selectedfrom Table 1. In some cases, the agent is an EV uptake inhibitor incombination with an antibody that binds to an inflammasome component. Insome cases, the EV uptake inhibitor is a heparin. In some cases, theheparin is Enoxaparin. In some cases, the antibody that binds to aninflammasome component is an antibody that specifically binds to acomponent of a mammalian AIM2, NLRP1, NLRP2, NLRP3 or NLRC4inflammasome. In some cases, the inflammasome component is caspase-1,ASC or AIM2. In some cases, the inflammasome component is ASC. In somecases, the antibody binds to the PYD, CARD domain or an epitope derivedfrom the PYD or CARD domain of the ASC protein. In some cases, theantibody binds to a protein having at least 85% sequence identity withan amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2. In some cases, the antibody inhibits ASC activity inthe lungs of the patient. In some cases, the composition is formulatedwith a pharmaceutically acceptable carrier or diluent. In some cases,the composition is administered intracerebroventricularly,intraperitoneally, intravenously or by inhalation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1N illustrateinflammasome activation in C57/BL6 mouse corticaland lung tissue post-TBI. FIG. 1A shows a representative immunoblot ofactive caspase-1, ASC, IL-18, IL-β, HMGB1, and AIM2 after TBI. Activecaspase-1 (FIG. 1B), ASC (FIG. 1C), IL-18 (FIG. 1D), HMGB1 (FIG. 1E),AIM 2 (FIG. 1F), and IL-β, (FIG. 1G), are significantly elevated incortical tissue at 4 and 24 h post-TBI. Data presented as mean+/- SEM;****p<0.001, ***p<0.01, p<0.05 compared to sham. N=4-5 per group. FIG.1H shows a representative immunoblot of active caspase-1, ASC, IL-18,IL-β, HMGB1, and AIM2 in lung tissue. I, J, K, L, M, N) Active caspase-1(FIG. 1I), ASC (FIG. 1J), IL-18 (FIG. 1K), HMGB1 (FIG. 1L), AIM 2 (FIG.1M), and IL-β, (FIG. 1N) are significantly elevated in lung tissue 4 and24 h after TBI. Data presented as mean+/-SEM. N= 4-5 per group,**p<0.01., *p<0.05 compared to sham.

FIGS. 2A-2C illustrates Expression of inflammasome proteins in Type IIalveolar epithelial cells. FIG. 2A shows AIM2, FIG. 2B shows activeCaspase-1 and FIG. 2C shows ASC immunoreactivity increases in lungtissue after CCI (4, 24 h) when compared to mice. Confocal images ofAIM2, caspase-1, and ASC (green) and type II epithelial cells(surfactant protein C, red).

FIGS. 3A-3E illustrates TBI increases nuclear and cytoplasmic HMGB1expression in mice lung. FIG. 3A shows representative immunoblot ofnuclear HMGB1 after TBI. FIG. 3B shows nuclear HMGB1 is significantlyelevated in 4 hour injured animals compared to sham. FIG. 3C showsrepresentative immunoblot of cytoplasmic HMGB1 after TBI. FIG. 3D showscytoplasmic HMGB1 is significantly elevated in 4 hour injured animalscompared to sham. Data presented as mean+/- SEM; ****p<0.001, ***p<0.01,*p<0.05 compared to sham. N=4-5 per group. FIG. 3E shows HMGB1immunoreactivity increased in lung tissue after CCI when compared tosham mice. Confocal images of HMGB1 and type II epithelial cells(surfactant protein C, red)

FIGS. 4A-4C illustrates Pyroptosome formation in mice lungs 4 hourspost-TBI. FIG. 4A shows TBI induces laddering of ASC in lung tissue,indicating formation of the pyroptosome, an oligomerization of ASCdimers that leads to activation of caspase-1 and pyroptosis. FIG. 4Bshows representative immunoblot and FIG. 4C shows quantification ofgasdermin. Gasdermin-D is significantly elevated in lung tissuepost-TBI. Data presented as mean+/-SEM. N= 4-5per group, **p<0.01.,*p<0.05 compared to sham.

FIGS. 5A-5B illustrates TBI induces alveolar morphological changes andacute lung injury in mice. FIG. 5A shows H&E staining of lung sectionsfrom sham and injured animals at 4 h and 24 h. Sections show evidence ofneutrophil infiltration (arrow heads), changes in morphology of alveolarcapillary membranes (asterisk, *), interstitial edema (short arrows),and evidence of thickening of the interstitium and the alveolar septum(pound, #). FIG. 5B shows acute lung injury scoring is significantlyincreased in injured animals when compared to sham at 4 h and 24 h. Datapresented as mean+/-SEM. N= 4-5 per group, **p<0.01., *p<0.05 comparedto sham.

FIG. 6 illustrates expression of CD81 in serum-derived EV from controland TBI-injured mice. Representative immunoblot of CD81 in serum-derivedEV from sham control and TBI-injured mice.

FIGS. 7A-7H illustrates adoptive transfer of EV from TBI animals inducecaspase-1 and ASC in the lungs of uninjured mice. FIG. 7A illustrates arepresentative immunoblot showing that caspase-1 (FIG. 7B), ASC (FIG.7C), IL-18 (FIG. 7D), AIM2 (FIG. 7E), HMGB1 (FIG. 7F) are elevated inthe lungs of animals that received EV isolated from TBI mice whencompared to EV from sham animals. Data presented as mean+/- SEM;*p<.0.05 compared to sham. N=3 per group. EV from TBI mice inducedalveolar morphological changes (decreased alveolar size) andinfiltration of inflammatory cells as determined by H&E staining (FIG.7G). ALI score is significantly increased in EV delivered from injuredmice compared to uninjured mice (FIG. 7H). Data presented as mean+/-SEM; *p<.0.05 compared to uninjured group.

FIGS. 8A-8F illustrates treatment with Enoxaparin (3 mg/kg) and IC 100(5 mg/kg) reduces inflammasome expression in lungs of animals deliveredEV from injured mice. FIG. 8A illustrates a representative immunoblotshowing that caspase-1 (FIG. 8B), ASC (FIG. 8C), IL-1β (FIG. 8D), AIM2(FIG. 8E), HMGB1 (FIG. 8F) are reduced in the lungs of animals that weretreated with Enoxaparin and IC 100 when compared to untreated positivecontrol animals. Data presented as mean+/- SEM; *p<.0.05 compared tosham. N=4 per group.

FIGS. 9A-9E illustrates treatment with Enoxaparin (3 mg/kg) and IC 100(5 mg/kg) reduces ALI score in lungs of animals delivered EV frominjured mice. FIGS. 9A-9D illustrates H&E staining of lung sections fromsaline (FIG. 9A), untreated (FIG. 9B), Enoxaparin (FIG. 9C) and IC 100(Anti-ASC; FIG. 9D) treated mice lungs delivered EV from injuredanimals. Sections show evidence of neutrophil infiltration, changes inmorphology of alveolar capillary membranes, interstitial edema, andevidence of thickening of the interstitium and the alveolar septum. FIG.9E illustrates that acute lung injury scoring is significantly decreasedin animals treated with Enoxaparin, IC 100 when compared to untreatedanimals. Data presented as mean+/-SEM. N= 4 per group, **p<0.01.,*p<0.05.

FIGS. 10A-10F illustrates delivery of serum-derived EV from TBI patientsincreases inflammasome protein expression in pulmonary endothelialcells. FIG. 10A shows western blot representation of caspase-1, ASC,AIM2, HMGB1 in PMVEC after incubation with TBI-EV and control-EV for 4hours. FIGS. 10B-10E) shows quantification of western blots, n=3 filtersper group, n=6 patients, t-test, p<0.05. FIG. 10F shows immunoassayresults of a significant increase in IL-1β expression using Ella simpleplex assay n=3 filters per group, n=6 patients, t-test, p<0.05.

FIGS. 11A-11C illustrates delivery of TBI-EV to pulmonary endothelialcells increases immunoreactivity of active caspase-1 and cell death.FIG. 11A shows co-localization of Caspase-1 FLICA and PI staining andPMVEC incubated with TBI-EV for 4 hours. FIG. 11B shows caspsae-1 FLICAand PI staining in PMVEC incubated with control-EV for 4 hours. FIG. 11Cshows fluorescent plate reader analysis of PMVEC incubated with TBI andcontrol-EV for 4 hours. n=6, p<0.05.

DETAILED DESCRIPTION Definitions

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.

As used herein, the term “antibody” refers generally and broadly toimmunoglobulins, monoclonal antibodies, and polyclonal antibodies, aswell as active fragments thereof. The fragment may be active in that itbinds to the cognate antigen (e.g., ASC, NLRP1, AIM2, etc.), or it maybe active in that it is biologically functional. The antibodies for useherein may be chimeric, humanized, or human, using techniques known inthe art.

As used herein, the term “humanized antibody” refers to an antibody inwhich minimal portions of a non-human antibody are introduced into anotherwise human antibody.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein is substantially non-immunogenicin humans, with only minor sequence changes or variations.

An antigen binding site can be generally formed by the heavy chainvariable region (VH) and the light chain variable region (VL)immunoglobulin domains, with the antigen-binding interface formed by sixsurface polypeptide loops, termed complimentarity determining regions(CDRs). There are three CDRs each in VH (HCDR1, HCDR2, HCDR3) and VL(LCDR1, LCDR2, LCDR3), together with framework regions (FRs).

The term “CDR region” or “CDR” can be mean the hypervariable regions ofthe heavy or light chains of the immunoglobulin as defined by Kabat etal., 1991 (Kabat, E. A. et al., (1991) Sequences of Proteins ofImmunological Interest, 5th Edition. US Department of Health and HumanServices, Public Service, NIH, Washington), and later editions. Anantibody typically contains 3 heavy chain CDRs and 3 light chain CDRs.

It has been shown that fragments of a whole antibody can also bindantigens. Examples of binding fragments include: (i) an Fab fragmentconsisting of VL, VH, CL and CH1 domains (Ward, E. S. et al., (1989)Nature 341, 544-546); (ii) an Fd fragment consisting of the VH and CH1domains (McCafferty et al., (1990) Nature, 348, 552-554); (iii) an Fvfragment consisting of the VL and VH domains of a single antibody (Holtet al., (2003) Trends in Biotechnology 21, 484-490); (iv) a dAb fragment(Ward, E. S. et al., Nature 341, 544-546 (1989), McCafferty, et al.,(1990) Nature, 348, 552-554, Holt et al., (2003) Trends in Biotechnology21, 484-490], which consists of a VH or a VL domain; (v) isolated CDRregions; (vi) F(ab′)₂ fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal., (1988) Science, 242, 423-426, Huston et al., (1988) PNAS USA, 85,5879-5883); (viii) bispecific single chain Fv dimers (PCT/US92109965)and (ix) “diabodies”, multivalent or multispecific fragments constructedby gene fusion (WO94/13804; Holliger, P. (1993) et al., Proc. Natl.Acad. Sci. USA 90 6444-6448).

Fv, scFv or diabody molecules may be stabilized by incorporation ofdisulfide bridges linking the VH and VL domains (Reiter, Y. et al.,Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFvjoined to a CH3 domain may also be made (Hu, S. et al., (1996) CancerRes., 56, 3055-3061). Other examples of binding fragments can be Fab′,which differs from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH1 domain, including one ormore cysteines from the antibody hinge region, and Fab′-SH, which is aFab′ fragment in which the cysteine residue(s) of the constant domainsbear a free thiol group.

“Fv” when used herein can refer to the minimum fragment of an antibodythat retains both antigen-recognition and antigen-binding sites. “Fab”when used herein can refer to a fragment of an antibody that comprisesthe constant domain of the light chain and the CH1 domain of the heavychain. The term “mAb” refers to monoclonal antibody.

By the terms “Apoptosis-associated Speck-like protein containing aCaspase Activating Recruitment Domain (CARD)” and “ASC” is meant anexpression product of an ASC gene or isoforms thereof, or a protein thatshares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or99%) amino acid sequence identity with ASC (e.g., NP_037390 (Q9ULZ3-1),NP_660183 (Q9ULZ3-2) or Q9ULZ3-3 in human, NP 075747 in mouse orNP_758825 (BAC43754) in rat) and displays a functional activity of ASC.A “functional activity” of a protein is any activity associated with thephysiological function of the protein. Functional activities of ASCinclude, for example, recruitment of proteins for activation ofcaspase-1 and initiation of cell death.

By the term “ASC gene,” or “ASC nucleic acid” is meant a nativeASC-encoding nucleic acid sequence, genomic sequences from which ASCcDNA can be transcribed, and/or allelic variants and homologues of theforegoing. The terms encompass double-stranded DNA, single-stranded DNA,and RNA.

As used herein, the term “inflammasome” means a multi-protein (e.g., atleast two proteins) complex that activates caspase-1. Further, the term“inflammasome” can refer to a multi-protein complex that activatescaspase-1 activity, which in turn regulates IL-1β, IL-18 and IL-33processing and activation. See Arend et al. 2008; Li et al. 2008; andMartinon et al. 2002, each of which is incorporated by reference intheir entireties. The terms “NLRP1 inflammasome”, “NALP1 inflammasome”,“NLRP2 inflammasome”, “NALP2 inflammasome”, “NLRP3 inflammasome”, “NALP3inflammasome”, “NLRC4 inflammasome”, “IPAF inflammasome” or “AIM2inflammasome” mean a protein complex of at least caspase-1 and oneadaptor protein, e.g., ASC. For example, the terms “NLRP1 inflammasome”and “NALP1 inflammasome” can mean a multiprotein complex containingNLRP1, ASC, caspase-1, caspase-11, XIAP, and pannexin-1 for activationof caspase-1 and processing of interleukin-1β, interleukin-18 andinterleukin-33. The terms “NLRP2 inflammasome” and “NALP2 inflammasome”can mean a multiprotein complex containing NLRP2 (aka NALP2), ASC andcaspase-1,while the terms “NLRP3 inflammasome” and “NALP3 inflammasome”can mean a multiprotein complex containing NLRP3 (aka NALP3), ASC andthe terms “NLRC4 inflammasome” and “IPAF inflammasome” can mean amultiprotein complex containing NLRC4 (aka IPAF), ASC and caspase-1.Additionally, the term “AIM2 Inflammasome” can mean a multiproteincomplex comprising AIM2, ASC and caspase-1.

As used herein, the phrase “sequence identity” means the percentage ofidentical subunits at corresponding positions in two sequences (e.g.,nucleic acid sequences, amino acid sequences) when the two sequences arealigned to maximize subunit matching, i.e., taking into account gaps andinsertions. Sequence identity can be measured using sequence analysissoftware (e.g., Sequence Analysis Software Package from Accelrys CGC,San Diego, CA).

By the phrases “therapeutically effective amount” and “effective dosage”is meant an amount sufficient to produce a therapeutically (e.g.,clinically) desirable result; the exact nature of the result will varydepending on the nature of the disorder being treated. For example,where the disorder to be treated is SCI, the result can be animprovement in motor skills and locomotor function, a decreased spinalcord lesion, etc. The compositions described herein can be administeredfrom one or more times per day to one or more times per week. Theskilled artisan will appreciate that certain factors can influence thedosage and timing required to effectively treat a subject, including butnot limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the compositions of the inventioncan include a single treatment or a series of treatments.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent described herein, or identified bya method described herein, to a patient, or application oradministration of the therapeutic agent to an isolated tissue or cellline from a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease, or the predisposition toward disease.

The terms “patient” “subject” and “individual” are used interchangeablyherein, and mean a mammalian subject to be treated, with human patientsbeing preferred. In some cases, the methods of the invention find use inexperimental animals, in veterinary applications, and in the developmentof animal models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters, as well as primates.

As interchangeably used herein, “Absent in Melanoma 2” and “AIM2” canmean an expression product of an AIM2 gene or isoforms; or a proteinthat shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98,or 99%) amino acid sequence identity with AIM2 (e.g., accessionnumber(s) NX 014862, NP004824, XP016858337, XP005245673, AAB81613,BAF84731, AAH10940) and displays a functional activity of AIM2.

As interchangeably used herein, “NALP1” and “NLRP1” mean an expressionproduct of an NALP1 or NLRP1 gene or isoforms; or a protein that sharesat least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%)amino acid sequence identity with NALP1 (e.g., accession number(s)AAH51787, NP_001028225, NP_127500, NP_127499, NP_127497, NP055737) anddisplays a functional activity of NALP1.

As interchangeably used herein, “NALP2” and “NLRP2” mean an expressionproduct of an NALP2 or NLRP2 gene or isoforms; or a protein that sharesat least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%)amino acid sequence identity with NALP2 (e.g., accession number(s)NP_001167552, NP_001167553, NP_001167554 or NP_060322) and displays afunctional activity of NALP2.

As interchangeably used herein, “NALP3” and “NLRP3” mean an expressionproduct of an NALP3 or NLRP3 gene or isoforms; or a protein that sharesat least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%)amino acid sequence identity with NALP3 (e.g., accession number(s)NP_001073289, NP_001120933, NP_001120934, NP_001230062, NP_004886,NP_899632, XP_011542350, XP_016855670, XP_016855671, XP_016855672 orXP_016855673) and displays a functional activity of NALP3.

As interchangeably used herein, “NLRC4” and “IPAF” mean an expressionproduct of an NLRC4 or IPAF gene or isoforms; or a protein that sharesat least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%)amino acid sequence identity with NLRC4 (e.g., accession number(s)NP_001186067, NP001186068, NP_001289433 or NP_067032) and displays afunctional activity of NLRC4.

By the terms “stroke” and “ischemic stroke” is meant when blood flow isinterrupted to part of the brain or spinal cord.

By “traumatic injury to the CNS” is meant any insult to the CNS from anexternal mechanical force, possibly leading to permanent or temporaryimpairments of CNS function.

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies,anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled insoluble or bound form, as well as fragments, regions or derivativesthereof, provided by any known technique, such as, but not limited to,enzymatic cleavage, peptide synthesis or recombinant techniques. Suchanti-ASC and anti-NLHP1 antibodies of the present invention are capableof binding portions of ASC and NLRP1, respectively,that interfere withcaspase-1 activation.

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates).Immunology techniques are generally known in the art and are describedin detail in methodology treatises such as Advances in Immunology,volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA, 2007;Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howardand Matthew R. Kaser, CRC Press, Boca Raton, FL, 2006; MedicalImmunology, 6^(th) ed., edited by Gabriel Virella, Informa HealthcarePress, London, England, 2007; and Harlow and Lane ANTIBODIES: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY, 1988.

Although compositions and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable compositions and methods are described below. Allpublications, patent applications, and patents mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Theparticular embodiments discussed below are illustrative only and notintended to be limiting.

OVERVIEW

Provided herein are compositions and methods for reducing inflammationin the lungs of a mammal that has been subjected to or is afflicted by acondition that results in or causes lung inflammation. The compositionsand methods described herein can include antibodies or active fragmentsthereof as provided herein that specifically bind to at least onecomponent (e.g., ASC) of a mammalian inflammasome and/or compounds thatmodulate (e.g., inhibit or reduce) extracellular vesicle (EV) uptake andhave use as treatments for lung inflammation in a mammal.

Described herein are methods for reducing inflammation in the lungs of amammal having a condition that results in and/or causes an inflammatoryresponse in the lungs. In one embodiment, the method of treatinginflammation in the lungs of a mammal comprises administering to themammal a composition comprising an agent that inhibits inflammasomesignaling. The mammal can be a patient or subject as provided herein.Examples of conditions that can lead to inflammation in the lungsinclude a central nervous system (CNS) injury (e.g., spinal cord injury(SCI), traumatic brain injury (TBI) or stroke), a neurodegenerativedisease, an autoimmune disease, asthma, chronic obstructive pulmonarydisease (COPD), cystic fibrosis, interstitial lung disease or acuterespiratory distress syndrome. The composition can be administered in atherapeutically effective amount. The therapeutically effective amountcan be a dose as provided herein. The agent can be an extracellularvesicle (EV) uptake inhibitor, an antibody or an active fragment thereofas provided herein that binds to a component of an inflammasome or acombination thereof. The composition can be administered by any suitableroute, e.g., by inhalation, intravenously, intraperitoneally, orintracerebroventricularly. The composition can further include at leastone pharmaceutically acceptable carrier or diluent.

In one embodiment, administration of the agent can result in a reductionin the activity and/or expression level of a component of a mammalianinflammasome in the lungs of the subject. The reduction can be in cellsof the lung such as, for example, Type II alveolar cells. The reductioncan be in comparison to a control. The control can be the subject priorto administration of the agent. The control can be the activity and/orexpression level of the inflammasome component(s) in a subject notadministered the agent. In one embodiment, administration of the agentresults in the reduction of caspase-1 activation in at least the lungsor lung cells of the subject. In one embodiment, administration of theagent results in the reduction of the expression level of one or moreinflammasome components (e.g., ASC, AIM2, NALP1, NALP2, NALP2, NALP3 orNLRC4) in at least the lungs or lung cells of the subject.

In another embodiment, administration of the agent can result in areduction in or elimination of acute lung injury (ALI). In oneembodiment, the reduction in ALI is evidenced by a reduction inneutrophil infiltration into alveolar and/or interstitial space, reducedor absent alveolar septal thickening or a combination thereof. Thereduction can be in comparison to a control. The control can be ALI inthe subject prior to administration of the agent. The control can be ALlin a subject suffering from ALI not administered the agent.

In still another embodiment, administration of the agent can result in areduction in or elimination of pyroptosis in the lungs of the subject.Pyroptosis is a proinflammatory form of cell death that involvesactivation of caspase-1. Pyroptosis can be triggered by the caspase-1mediated cleavage of gasdermin D (GSDMD). In one embodiment, thereduction in pyroptosis is evidenced by a reduction in or lack ofcleavage of GSDMD in the lungs or lung cells (e.g., Type II alveolarcells) of the subject. The reduction or elimination of pyroptosis can bein comparison to a control. The reduction in or lack of cleavage ofGSDMD can be in comparison to a control. The control can be the level ofpyroptosis in the subject prior to administration of the agent. Thecontrol can be the level of pyroptosis in a subject suffering frompyroptosis not administered the agent.

In one embodiment, the agent to be administered is an EV uptakeinhibitor. The EV uptake inhibitor can be a compound, antisense RNA,siRNA, peptide, antibody or an active fragment thereof as providedherein or a combination thereof. The compound or peptide can be one ormore compounds selected from heparin, α-difluoromethylornithine (DFMO),Enoxaparin, Asialofetuin, Human receptor-associated protein (RAP), RGD(Arg-Gly-Asp) peptide, Cytochalasin D, Cytochalasin B,Ethylenediaminetetra acetic acid (EDTA), Latrunculin A, Latrunculin B,NSC23766, Dynasore, Chlorpromazine, 5-(N-Ethyl-N-isopropyl)amiloride(EIPA), Amiloride, Bafilomycin A Monensin and Chloroquine, Annexin-V,Wortmannin, LY294002, Methyl-β-cyclodextrin (MβCD), Filipin,Simvastatin, Fumonisin B1 and N-butyldeoxynojirimycin hydrochloride,U0126 or a proton pump inhibitor. The EV uptake inhibitor antibody or anactive fragment thereof as provided herein can be one or more antibodiesor active fragments thereof directed against protein targets listed inTable 1. A composition for treating and/or reducing inflammation in thelungs of a mammal using an EV uptake inhibitor can further include atleast one pharmaceutically acceptable carrier or diluent.

TABLE 1 Exemplary targets and corresponding antibodies for use inblocking EV uptake. Gene Symbol Gene Name Exemplary Antibodies ICAM-1Intercellular Adhesion Molecule 1 Invitrogen ICAM-1 antibody (LifeTechnologies, 07-5403); CD54 (ICAM-1) Monoclonal Antibody (R6.5),eBioscience™ LFA-1 Lymphocyte function-associated antigen 1 AbbiotecLFA-1 antibody (Abbiotec, 250944); Developmental Studies Hybridoma BankLFA-1 antibody (Developmental Studies Hybridoma Bank, MHM24) TIM-4T-cell membrane protein 4 BioLegend TIMD4 antibody (BioLegend, 354004);LifeSpan Biosciences TTMD4 antibody (Lifespan Biosciences, LS-B1413)MFG-E8 Milk Fat Globule-EGF Factor 8 Protein MBL International MFGE8antibody (MBL, D199-3); Santa Cruz Biotechnology MFGE8 antibody (SantaCruz, sc-8029); MBL International MFGE8 antibody (MBL, 18A2-G10) DC-SIGNDendritic Cell-Specific Intercellular adhesion molecule-3-GrabbingNon-integrin Invitrogen DC SIGN antibody (eBioscience, eB-h209,17-2099-41); BD Biosciences DC SIGN antibody (BD, DCN46, 551186) DEC205cluster of differentiation 205 EMD Millipore LY75 antibody (Millipore,HD30); BioLegend LY75 antibody (BioLegend, 342203) H-2Kb MHC Class I(H-2Kd) BioLegend H2-K1 antibody (BioLegend, 28-8-6, 114603); BioLegendH2-K1 antibody (BioLegend, 28-14-8, 14-5999-85) Tspan8 Tetraspanin-8 Rand D Systems TSPAN8 antibody (R&D Systems, MAB4734) Tspan29Tetraspanin-29 Santa Cruz Biotechnology CD9 antibody (Santa Cruz,sc-59140); Invitrogen CD9 antibody (eBioscience, eBioSN4; BD BiosciencesCD9 antibody (BD Pharmingen, 555370) ITGAL Integrin subunit alpha LTS1/22.1.1.13.3;M17/4.4.11.9 ITGAM Integrin subunit alpha M CD11bMonoclonal Antibody (VIM12)(CD11B00); BD Biosciences CD11b antibody (BDPharmingen, ICRF44; 555385) ITGAX Integrin subunit alpha X Anti-IntegrinαX Antibody, clone N418 (MAB1399Z); BD Biosciences CD11c antibody (BDBioscience, B-ly6; 560369) CD44 Cluster of differentiation 44 InvitrogenCD44 antibody (eBioscience, VFF-7; MA1-82392); Invitrogen CD44 antibody(eBioscience, IM7; MA1-10225); Invitrogen CD44 antibody (eBioscience,5F12; MA5-12394); BD Biosciences CD44 antibody (BD Biosciences, 515;550990 OR 550988) ITGA3 Integrin subunit alpha 3 EMD Millipore integrinalpha3 antibody (Millipore, P1B5; MAB1952Z OR MAB1952P) ITGA4 Integrinsubunit alpha 4 Bio X Cell ITGA4 antibody (BioXcell, PS/2) (BE0071-5MG);BD Biosciences ITGA4 antibody (BD Biosciences, 561892); BD BiosciencesITGA4 antibody (BD, 340976); EMD Millipore ITGA4 antibody (Millipore,P4C2; MAB1955) ITGAV Integrin subunit alpha V Abcam integrin alpha vantibody (Abcam, ab77906); Abeam integrin alpha v antibody (Abcam,ab78289); Abeam integrin alpha v antibody (Abeam, ab16821); Invitrogenintegrin alpha v antibody (Thermo Fisher Scientific, 272-17E6,MA1-91669); R & D Systems integrin alpha v antibody (R&D Systems,MAB2528) ITGB3 Integrin subunit beta 3 Abeam integrin beta3 antibody(Abcam, ab78289); Abnova integrin beta3 antibody (Abnova, MHF4, MAB7098)SELL Selectin L BioLegend CD62L antibody (Biolegend, 304804); BioLegendCD62L antibody (Biolegend, 304810) CD81 CD81 molecule BD BiosciencesCD81 antibody (BD Pharmingen, 555675); R and D Systems CD81 antibody(R&D Systems, MAB4615) LRP1 LDL receptor related protein 1 InvitrogenLRP1 antibody (Life Technologies, 37-7600); Invitrogen LRP1 antibody(Thermo Fisher, MA1-27198) VCAM1 vascular cell adhesion molecule 1Invitrogen VCAM-1 antibody (Caltag, IG11B1; MA5-16429); Immunotechanti-VCAM-1 antibody CD151 CD151 molecule (Raph blood group) BDBiosciences CD151 antibody (Becton Dickinson, 556056); Epitomics CD151antibody (Epitomics, 5901-1)

In one embodiment, the agent to be administered is an antibody or anactive fragment thereof as provided herein directed against a componentof a mammalian inflammasome or an antigen or epitope derived therefrom.In another embodiment, the agent to be administered is an antisense RNAor siRNA directed against a component of a mammalian inflammasome. Theinflammasome component can be a component of any inflammasome known inthe art, such as, for example, the NAPL1, NALP2, NALP3, NLRC4 or AIM2inflammasome. In a typical embodiment, the antibody specifically bindsto ASC or an antigen or epitope derived therefrom. However, an antibodyagainst any other component of a mammalian inflammasome (e.g., theNALP1, NALP2, NALP3, NLRC4 or AIM2 inflammasome) may be used.

An antibody as described herein can be a monoclonal or polyclonalantibody or active fragments thereof. Said antibodies or activefragments can be chimeric, human or humanized as described herein.

Any suitable antibody or an active fragment thereof as provided hereinthat specifically binds ASC can be used, e.g., an antibody that inhibitsASC activity in lung cells (e.g., Type II alveolar cells) of thesubject. In a typical embodiment, the antibody specifically binds to anamino acid sequence having at least 85% sequence identity with aminoacid sequence SEQ ID NO:1 or SEQ ID NO:2. Similarly, in anotherembodiment, the inflammasome is the NALP1 inflammasome, and the at leastone component is NALP1 (i.e., NLRP1). In this embodiment, the antibodyor an active fragment thereof as provided herein specifically binds toan amino acid sequence having at least 85% sequence identity with aminoacid sequence SEQ ID NO: 3 or SEQ ID NO: 4.

In yet another embodiment, the agent is one or more EV uptake inhibitorsin combination with one or more antibodies or active fragments thereofas provided herein that bind a component of an inflammasome. The EVuptake inhibitor can be any EV uptake inhibitor as provided herein. Theantibody that binds a component of an inflammasome can any antibody thatbinds any inflammasome component as provided herein. In one embodiment,the agent administered to a subject suffering from lung inflammationcomprises a heparin (e.g., Enoxaparin) in combination with an antibodythat binds a component of the AIM2 inflammasome (e.g., ASC).

In one embodiment, the method comprises: providing a therapeuticallyeffective amount of a composition including an antibody or an activefragment thereof as provided herein that specifically binds to at leastone component (e.g., ASC) of a mammalian inflammasome (e.g., AIM2inflammasome); and administering the composition to the mammal sufferingfrom lung inflammation, wherein administering the composition to themammal results in a reduction of caspase-1 activation in the lungs ofthe mammal. In another embodiment, the method comprises: providing atherapeutically effective amount of a composition including an antibodythat specifically binds to at least one component (e.g., ASC) of amammalian inflammasome (e.g., AIM2 inflammasome); and administering thecomposition to the mammal suffering from lung inflammation, whereinadministering the composition to the mammal results in a reduction inthe levels of one or more inflammasome components (e.g., ASC). In yetanother embodiment, the method comprises: providing a therapeuticallyeffective amount of a composition including an antibody thatspecifically binds to at least one component (e.g., ASC) of a mammalianinflammasome (e.g., AIM2 inflammasome); and administering thecomposition to the mammal suffering from lung inflammation, whereinadministering the composition to the mammal results in a reduction ALI.The lung inflammation can be the result of a CNS injury (e.g., SCI orTBI), asthma, chronic obstructive pulmonary disorder (COPD), aneurodegenerative disease, or an autoimmune disease with an inflammatorycomponent In one embodiment, the lung inflammation is caused by a CNSinjury such as TBI or SCI.

In one embodiment, the methods provided herein further entail detectinga level or activity of one or more components of a mammalianinflammasome in a sample from a subject suspected of suffering from lunginflammation. The method of detecting the level or activity entailsmeasuring the level of at least one inflammasome protein (e.g., ASC orAIM2) in the sample obtained from the subject; determining the presenceor absence of an elevated level or activity of said at least oneinflammasome protein (e.g., ASC or AIM2). The level or activity of saidat least one inflammasome protein can be enhanced relative to the levelof said at least one inflammasome protein in a control sample. The levelor activity of said at least one inflammasome protein in the proteinsignature can be enhanced relative to a pre-determined reference valueor range of reference values. The at least one inflammasome protein canbe nucleotide-binding leucine-rich repeat pyrin domain containingprotein 1 (NLRP1), NLRP2, NLRP3, NLRC4, AIM2, apoptosis-associatedspeck-like protein containing a caspase recruitment domain (ASC),caspase-1, or combinations thereof. The sample can be cerebrospinalfluid (CSF), saliva, blood, serum, plasma, urine or a lung aspirate.

Antibodies That Bind Specifically to At Least One Component of AMammalian Inflammasome

The methods described herein for reducing inflammation in the lungs of amammal include compositions including an antibody or an active fragmentthereof as provided herein that specifically binds to at least onecomponent (e.g., ASC, AIM2) of a mammalian inflammasome (e.g., the AIM2inflammasome). A composition for treating and/or reducing inflammationin the lungs of a mammal can further include at least onepharmaceutically acceptable carrier or diluent. Exemplary antibodiesdirected against components of a mammalian inflammasome for use in themethods herein can be those found in US8685400, the contents of whichare herein incorporated by reference in its entirety.

In one embodiment, a composition for treating and/or reducinginflammation in the lungs of a mammal includes an antibody or an activefragment thereof as provided herein that specifically binds to a domainor portion thereof of a mammalian ASC protein such as, for example, ahuman, mouse or rat ASC protein. Any suitable anti-ASC antibody can beused, and several are commercially available. Examples of anti-ASCantibodies for use in the methods herein can be those found inUS8685400, the contents of which are herein incorporated by reference inits entirety. Examples of commercially available anti-ASC antibodies foruse in the methods provided herein include, but are not limited to04-147 Anti-ASC, clone 2EI-7 mouse monoclonal antibody fromMilliporeSigma, AB3607 - Anti-ASC Antibody from Millipore Sigma,orb194021 Anti-ASC from Biorbyt, LS-C331318-50 Anti-ASC from LifeSpanBiosciences, AF3805 Anti-ASC from R & D Systems, NBP1-78977 Anti-ASCfrom Novus Biologicals, 600-401-Y67 Anti-ASC from RocklandImmunochemicals, D086-3 Anti-ASC from MBL International, AL177 anti-ASCfrom Adipogen, monoclonal anti-ASC (clone o93E9) antibody, anti-ASCantibody (F-9) from Santa Cruz Biotechnology, anti-ASC antibody (B-3)from Santa Cruz Biotechnology, ASC polyclonal antibody – ADI-905-173from Enzo Life Sciences, or A161 Anti-Human ASC - Leinco Technologies.The human ASC protein can be accession number NP_037390.2 (Q9ULZ3-1), NP660183 (Q9ULZ3-2) or Q9ULZ3-3. The rat ASC protein can be accessionnumber NP 758825 (BAC43754). The mouse ASC protein can be accessionnumber NP_075747.3. In one embodiment, the antibody binds to aPYRIN-PAAD-DAPIN domain (PYD) or a portion or fragment thereof of amammalian ASC protein (e.g. human, mouse or rat ASC). In thisembodiment, an antibody as described herein specifically binds to anamino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%)sequence identity with a PYD domain or fragment thereof of human, mouseor rat ASC. In one embodiment, the antibody binds to a C-terminalcaspase-recruitment domain (CARD) or a portion or fragment thereof of amammalian ASC protein (e.g. human, mouse or rat ASC). In thisembodiment, an antibody as described herein specifically binds to anamino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%)sequence identity with a CARD domain or fragment thereof of human, mouseor rat ASC. In still another embodiment, the antibody binds to a portionor fragment thereof of a mammalian ASC protein sequence (e.g. human,mouse or rat ASC) located between the PYD and CARD domains. In anotherembodiment, a composition for treating and/or reducing inflammation inthe lungs of a mammal includes an antibody that specifically binds to aregion of rat ASC, e.g., amino acid sequence

ALRQTQPYLVTDLEQS (SEQ ID NO: 1)

(i.e., residues 178-193 of rat ASC, accession number BAC43754). In thisembodiment, an antibody as described herein specifically binds to anamino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%)sequence identity with amino acid sequence

ALRQTQPYLVTDLEQS (SEQ ID NO: 1)

of rat ASC. In another embodiment, a composition for treating and/orreducing inflammation in the CNS of a mammal includes an antibody thatspecifically binds to a region of human ASC, e.g., amino acid sequence

RESQSYLVEDLERS (SEQ ID NO:2).

In one embodiment, an antibody that binds to an ASC domain or fragmentthereof as described herein inhibits ASC activity in lung cells, e.g.,Type II alveolar cells of a mammal.

In another embodiment, a composition for reducing inflammation in thelungs of a mammal includes an antibody or an active fragment thereof asprovided herein that specifically binds to NLRP1 (e.g., anti-NLRP1chicken antibody) or a domain thereof. Any suitable anti-NLRP1 antibodycan be used, and several are commercially available. Examples ofanti-NLRP1 antibodies for use in the methods herein can be those foundin US8685400, the contents of which are herein incorporated by referencein its entirety. Examples of commercially available anti-NLRP1antibodies for use in the methods provided herein include, but are notlimited to human NLRP1 polyclonal antibody AF6788 from R&D Systems, EMDMillipore rabbit polyclonal anti-NLRP1 ABF22, Novus Biologicals rabbitpolyclonal anti-NLRP1 NB100-56148, Sigma-Aldrich mouse polyclonalanti-NLRP1 SAB1407151, Abcam rabbit polyclonal anti-NLRP1 ab3683,Biorbyt rabbit polyclonal anti-NLRP1 orb325922 mybiosource rabbitpolyclonal anti-NLRP1 MBS7001225, R&D systems sheep polyclonal AF6788,Aviva Systems mouse monoclonal anti-NLRP1 oaed00344, Aviva Systemsrabbit polyclonal anti-NLRP1 AR054478_P050, Origene rabbit polyclonalanti-NLRP1 APO7775PU-N, Antibodies online rabbit polyclonal anti-NLRP1ABIN768983, Prosci rabbit polyclonal anti-NLRP1 3037, Proteintech rabbitpolyclonal anti-NLRP1 12256-1-AP, Enzo mouse monoclonal anti-NLRP1ALX-804-803-C100, Invitrogen mouse monoclonal anti-NLRP1 MA1-25842,GeneTex mouse monoclonal anti-NLRP1 GTX16091, Rockland rabbit polyclonalanti-NLRP1 200-401-CX5, or Cell Signaling Technology rabbit polyclonalanti-NLRP1 4990. The human NLRP1 protein can be accession numberAAH51787, NP_001028225, NP_055737, NP_127497, NP_127499, or NP_127500.In one embodiment, the antibody binds to a Pyrin, NACHT, LRR1-6, FIINDor CARD domain or a portion or fragment thereof of a mammalian NLRP1protein (e.g. human NLRP1). In this embodiment, an antibody as describedherein specifically binds to an amino acid sequence having at least 65%(e.g., 65, 70, 75, 80, 85%) sequence identity with a specific domain(e.g., Pyrin, NACHT, LRR1-6, FIIND or CARD) or fragment thereof of humanNLRP1. In one embodiment, a chicken anti-NLRP1 polyclonal that wascustom-designed and produced by Ayes Laboratories is used for reducinglung inflammation. This antibody can be directed against the followingamino acid sequence in human NLRP1:

CEYYTEIREREREKSEKGR (SEQ ID NO:3).

In one embodiment, an antibody that binds to a NLRP1 domain or fragmentthereof as described herein inhibits NLRP1 activity in lung cells, e.g.,Type II alveolar cells of a mammal.

In yet another embodiment, a composition for reducing inflammation inthe lungs of a mammal includes an antibody or an active fragment thereofas provided herein that specifically binds to AIM2 or a domain thereof.Any suitable anti-AIM2 antibody can be used, and several arecommercially available. Examples of commercially available anti-AIM2antibodies for use in the methods provided herein include, but are notlimited to a rabbit polyclonal anti-AIM2 cat. Number 20590-1-AP fromProteintech,, Abcam anti-AIMS antibody (ab119791), rabbit polyclonalanti-AIM2 (N-terminal region) Cat. Number AP3851 from ECM biosciences,rabbit polyclonal anti-ASC Cat. Number E-AB-30449 from Elabsciences,,Anti-AIM2 mouse monoclonal antibody called AIM2 Antibody (3C4G11) withcatalog number sc-293174 from Santa Cruz Biotechnology, mouse monoclonalAIM2 antibody with catalog number TA324972 from Origene, AIM2 monoclonalantibody (10M2B3) from Thermofisher Scientific, AIM2 rabbit polyclonalantibody ABIN928372 or ABIN760766 from Antibodies-online, Biomatix coatanti-AIM2 polyclonal antibody with cat. Number CAE02153. Anti-AIM2polyclaonl antibody (OABF01632) from Aviva Systems Biology, rabbitpolyclonal anti-AIM2 antibody LS-C354127 from LSBio-C354127, rabbitmonoclonal anti-AIM2 antibody from Cell Signaling Technology, with catnumber MA5-16259. Rabbit polyclonal anti-AIM2 monoclonal antibody fromFab Gennix International Incorporated, Cat. Number AIM2 201AP,MyBiosource rabbit polyclonal anti-AIM2 cat number MBS855320, Signalwayrabbit polyclonal anti AIM2 cateaog number 36253, Novus Biologicalrabbit polyclonal anti-AIM2 catalog number 43900002, GeneTex rabbitpolclonal anti-AIM2 GTX54910, Prosci, rabbit polyclonal anti-AIM226-540, Biorbyt mouse monoclonal anti-AIM2 orb333902, Abcam rabbitpolyclonal anti-AIM2 ab93015), Abcam rabbit polyclonal anti-AIM2ab76423, Signma Aldrich mouse polyclonal anti-AIM2 SAB1406827, orBiolegend anti-AIM2 3B10. The human AIM2 protein can be accession numberNX_014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731 orAAH10940. In one embodiment, the antibody binds to a Pyrin or HIN-200domain or a portion or fragment thereof of a mammalian AIM2 protein(e.g. human AIM2). In this embodiment, an antibody as described hereinspecifically binds to an amino acid sequence having at least 65% (e.g.,65, 70, 75, 80, 85%) sequence identity with a specific domain (e.g.,Pyrin or HIN-200) or fragment thereof of human AIM2. In one embodiment,an antibody that binds to a AIM2 domain or fragment thereof as describedherein inhibits AIM2 activity in lung cells, e.g., Type II alveolarcells of a mammal.

Anti-inflammasome (e.g., Anti-ASC, anti-NLRP1 or anti-AIM2) antibodiesas described herein include polyclonal and monoclonal rodent antibodies,polyclonal and monoclonal human antibodies, or any portions thereof,having at least one antigen binding region of an immunoglobulin variableregion, which antibody specifically binds to a component of a mammalianinflammasome (e.g., AIM2 inflammasome) such as, for example, ASC orAIM2. In some cases, the antibody is specific for ASC such that anantibody is specific for ASC if it is produced against an epitope of thepolypeptide and binds to at least part of the natural or recombinantprotein.

Methods for determining monoclonal antibody specificity and affinity bycompetitive inhibition can be found in Harlow, et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988, Colligan et al., eds., Current Protocols inImmunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993), and Muller, Meth. Enzymol. 92:589-601, 1983, whichreferences are entirely incorporated herein by reference.

Anti-inflammasome (e.g., Anti-ASC and anti-AIM2) antibodies of thepresent invention can be routinely made according to methods such as,but not limited to inoculation of an appropriate animal with thepolypeptide or an antigenic fragment, in vitro stimulation of lymphocytepopulations, synthetic methods, hybridomas, and/or recombinant cellsexpressing nucleic acid encoding such anti-ASC or anti-NLR1 antibodies.Immunization of an animal using purified recombinant ASC or peptidefragments thereof, e.g., residues 178-193 (SEQ ID NO: 1) of rat ASC(e.g., accession number BAC43754) or SEQ ID NO:2 of human ASC, is anexample of a method of preparing anti-ASC antibodies. Similarly,immunization of an animal using purified recombinant NLRP1 or peptidefragments thereof, e.g., residues MEE SQS KEE SNT EG-cys (SEQ ID NO:4)of rat NALP1 or SEQ ID NO:3 of human NALP1, is an example of a method ofpreparing anti-NLRP1 antibodies.

Monoclonal antibodies that specifically bind ASC or NLRP1 may beobtained by methods known to those skilled in the art. See, for exampleKohler and Milstein, Nature 256:495-497, 1975; U.S. Pat. No. 4,376,110;Ausubel et al., eds., Current Protocols in Molecular Biology, GreenePublishing Assoc. and Wiley Interscience, N.Y., (1987, 1992); Harlow andLane ANTIBODIES: A Laboratory Manual Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 1988; Colligan et al., eds., CurrentProtocols in Immunology, Greene Publishing Assoc. and WileyInterscience, N.Y., (1992, 1993), the contents of which are incorporatedentirely herein by reference. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclassthereof A hybridoma producing a monoclonal antibody of the presentinvention may be cultivated in vitro, in situ or in vivo.

Administration of Compositions

The compositions of the invention may be administered to mammals (e.g.,rodents, humans) in any suitable formulation. For example, anti-ASCantibodies may be formulated in pharmaceutically acceptable carriers ordiluents such as physiological saline or a buffered salt solution.Suitable carriers and diluents can be selected on the basis of mode androute of administration and standard pharmaceutical practice. Adescription of exemplary pharmaceutically acceptable carriers anddiluents, as well as pharmaceutical formulations, can be found inRemington’s Pharmaceutical Sciences, a standard text in this field, andin USP/NF. Other substances may be added to the compositions tostabilize and/or preserve the compositions.

The compositions of the invention may be administered to mammals by anyconventional technique. Typically, such administration will be byinhalation or parenteral (e.g., intravenous, subcutaneous, intratumoral,intramuscular, intraperitoneal, or intrathecal introduction). Thecompositions may also be administered directly to a target site by, forexample, surgical delivery to an internal or external target site, or bycatheter to a site accessible by a blood vessel. The compositions may beadministered in a single bolus, multiple injections, or by continuousinfusion (e.g., intravenously, by peritoneal dialysis, pump infusion).For parenteral administration, the compositions are preferablyformulated in a sterilized pyrogen-free form.

Effective Doses

The compositions described above are preferably administered to a mammal(e.g., a rat, human) in an effective amount, that is, an amount capableof producing a desirable result in a treated mammal (e.g., reducinginflammation in the CNS of a mammal subjected to a traumatic injury tothe CNS or stroke or having an autoimmune or CNS disease). Such atherapeutically effective amount can be determined as described below.

Toxicity and therapeutic efficacy of the compositions utilized inmethods of the invention can be determined by standard pharmaceuticalprocedures, using either cells in culture or experimental animals todetermine the LD₅₀ (the dose lethal to 50% of the population). The doseratio between toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio LD₅₀/ED₅₀. Those compositions thatexhibit large therapeutic indices are preferred. While those thatexhibit toxic side effects may be used, care should be taken to design adelivery system that minimizes the potential damage of such sideeffects. The dosage of preferred compositions lies preferably within arange that includes an ED₅₀ with little or no toxicity. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration utilized.

As is well known in the medical and veterinary arts, dosage for any onesubject depends on many factors, including the subject’s size, bodysurface area, age, the particular composition to be administered, timeand route of administration, general health, and other drugs beingadministered concurrently.

EXAMPLES

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and should notbe construed as limiting the scope of the invention in any way.

Example 1: Role of EV Mediated Inflammasome Signaling in ALI FollowingTBI and Effects of its Neutralization

Pulmonary dysfunction often presents as a complication of SevereTraumatic Brain Injury (1). Approximately 20-25 percent of TBI subjectsdevelop acute lung injury (ALI) (2), but the mechanisms mediating thepathology of TBI-induced ALI remain poorly defined. Previous literaturehas supported the idea that pulmonary dysfunction after TBI is due tothe sympathetic response to increased intracranial pressure leading tocardiopulmonary dysfunction (42). More recent studies, however, haveshown that a systemic inflammatory response also plays a key role inTBI-induced lung injury (43). Specifically, the HMGB1-RAGE ligandreceptor pathway serves as central transduction mechanism for pulmonarydysfunction after TBI (8). In addition, HMGB1 induces AIM2 inflammasomeactivation (37). Furthermore previous literature reveals that pathogenssecrete EV that carry DAMPs, such as HMGB1, and trigger inflammation(Buzas et al., 2014). Various studies have shown that the blood brainbarrier (BBB) is permeable after TBI as early as 3-6 hours after injuryresulting in damage to the protective barrier between the brain and theintravascular compartment and leads to leakage of proteins and fluid(44). Disruption of the BBB after injury results in the secretion ofinflammatory mediators, such as DAMPs, which can further braininflammation and damage distal organs (5). Several inflammatorymediators can act as clear markers for brain injury, however theirvalidity is not widely accepted (45). Furthermore, there is currently noclinically approved treatment or biomarker for TBI-induced ALI.Recently, EV have become an area of interest in biomarker research for aseveral different types of diseases, including lung injury (46) and TBI(47). It has been previously shown that in EV isolated from thecerebrospinal fluid of patient with TBI, there is an increase ofinflammasome proteins when compared to control samples (14). In thisExample, the contribution of EV mediated inflammasome signaling in theetiology of TBI-induced ALI was examined.

Materials and Methods Animals and Traumatic Brain Injury

All animal procedures were approved by the Institutional Animal Care andUse Committee of the University of Miami Miller School of Medicine(Animal Welfare Assurance A3224-01) and were done according to the NIHGuide for the Care and Use of Laboratory Animals. The ARRIVE guidelineswere followed when conducting this study. All C57/BL6 mice were 8-12weeks and 24 to 32 grams. Mice were prospectively randomized toexperimental groups (sham, 4 h, 24) for TBI, experimental groups (naive,sham-saline, untreated, enoxaparin, anti-ASC) for adoptive transfer andtreatment.. For TBI experiment-groups, sham animals underwent surgicalprocedure but were not injured. For adoptive transfer treatment studies,the sham-saline group underwent surgical procedures and received salineas vehicle treatment. Naive animals underwent no surgical procedures. Asample size of 5 to 6 was used for each group based on power analysis(using G* power analysis, with an effect size F=0.85, α set a 0.05) andhistorical data ^(49,) ⁵⁰. All mice were housed in the viral antigenfree (VAF) animal facility at the Lois Pope Life Center at theUniversity of Miami on 12-hour light/dark cycles and food and water weresupplied ad libitum. The facility conducts husbandry procedures twice aweek and checks on the conditions of the animals daily. Animals wereobserved post-op, where they were kept on a heating pad and bodytemperature was controlled with a rectal probe where it was maintainedat 37° C., in our operation room and then transferred to the animalquarters.

Prior to surgery animals were anesthetized with ketamine and xylazine(intraperitoneal, i.p.). The anesthetized animals were then placed on aheating pad to ensure a body temperature of 37° C. TBI was performedusing a Controlled Cortical Impact (CCI) model. A 5 mm craniotomy wasmade on the right cortex (-2.5 mm posterior, 2.0 mm lateral fromBregma). Injury was induced using the ECCI-6.3 device (Custom Design &Fabrication, Richmond, VA, USA) with a 3 mm impounder at 6 m/s velocity,0.8 mm depth, and 150 ms impact duration (15). Following theseprocedures animals were returned to their cages and given food andwater. Animals were sacrificed at 4 hours and 24 hours after TBI asdescribed. Sham animals were anesthetized and subjected to the samepre-surgical incision as injured animals but did not undergo acraniotomy or contusion.

Tissue Collection

All animals were anesthetized with ketamine and xylazine, prior toperfusion. Animals then underwent tracheal perfusion. Lungs were infusedwith 4% paraformaldehyde (PFA) using a tracheal catheter at 20 cm H2Oand then fixed in 4% PFA overnight at 4° C. Fixed lung tissues wereparaffin embedded and 5 µm sections were processed (16). Right lungtissue was collected for protein isolation and molecular analyses.Animals then underwent decapitation and right cortical tissue wascollected for protein isolation and molecular analyses.

Pyroptosome Isolation Assay

Mice lung tissue lysates were filtered through a 5 µm low-bindingpolyvinylidene difluoride (PVDF) membrane (Millipore). After filtration,the supernatant was centrifuged at 2,700 xg for 8 minutes. The pelletwas resuspended in 40 µl of 3[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid (CHAPS) buffer (20 mmol/LHEPES-KOH, pH 7.5, 5 mmol/L MgCl2, 0.5 mmol/L EGTA, 0.1 mmol/Lphenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 0.1%CHAPS). The pyroptosome was pelleted by centrifugation at 2,700 xg for 8minutes. The pellet was then resuspended and incubated in 27.8 µl ofCHAPS buffer with 2.2 µl of disuccinimidyl substrate (9) for 30 minutesat room temperature to cross-link ASC dimers. Lastly, an equal amount of2× Laemmli buffer was added and proteins were analyzed by immunoblottingusing commercially available antibodies to ASC and Gasdermin D (GSD)..

Nuclear and Cytoplasmic Extraction

Nuclear and Cytoplasmic fractions were extracted using the NE-PERNuclear and Cytoplasmic Extraction Reagents (Thermo Scientific)according to manufacturer instructions. Briefly, mice lung tissuesamples were cut into 20-100 mg pieces and centrifuged at 500 × g for 5minutes. Tissue pieces were the homogenized with the CytoplasmicExtraction Reagent and centrifuged at 16,000 × g for 5 minutes. Then thesupernatant (cellular extract) was removed and the pellet wascentrifuged with Nuclear Extraction Reagent (Thermo Scientific) at16,000 × g for 10 minutes. This supernatant corresponded to the nuclearfraction, which was removed and stored at -80° C.

Immunoblotting

Lung and brain tissue samples were snap frozen in liquid nitrogen andstored in -80° C. 2-mm sections of right lower lung and right corticaltissue were homogenized in extraction buffer containing protease andphosphatase inhibitor cocktail (Sigma, St Louis, MO, USA) and resolvedin 4-20% Tris-TGX Criterion precasted gels (Bio-Rad, Hercules, CA, USA)as described in de Rivero Vaccari et al. 2015 (13) using antibodies tocaspase-1 (Novus Biologicals), ASC (Santa Cruz), IL-1 β (CellSignaling), IL-18 (Abcam) AIM2 (Santa Cruz) and HMGB1(Millipore).Quantification of band density was performed using Image Lab and alldata were normalized to β-actin.

Immunohistochemistry

Tissue sections were deparaffinized in xylene and then rehydrated usingethanol and Tris buffer saline. Immunohistochemical procedures were thencarried out for double staining as previously described (16). Sectionswere incubated overnight at 4° C. with antibodies against Caspase-1 andASC (Millipore), AIM2 (Santa Cruz), HMGB1(Millipore) and SPC(Millipore). Immunostained lung sections of sham, 4 hour, and 24 hourmice were examined with a Zeiss laser scanning confocal microscope(Zeiss, Inc., Thornwood, NY, USA). Lung sections were analyzed byindividuals who were blinded to the groups.

EV Isolation

EV were isolated from serum from TBI-injured mice and injury mice usingthe Total Exosome Isolation solution according to manufacturer’sinstructions (Invitrogen). Briefly, 100 µl of each sample werecentrifuged at 2000 × g for 30 minutes. The supernatant was thenincubated with 20 µl of Total Exosome Isolation (TEI) reagent for 30minutes at 4° C. followed by centrifugation at 10,000 × g for 10 minutesat room temperature. Supernatants were discarded and the pellet wasresuspended in 100 µl of PBS. EV were characterized by the expression ofCD81 and by Nanosight tracking analysis (FIG. 6 ).

Adoptive Transfer of EV

Serum-derived EV from C57BL-6 TBI and sham mice were injected into naïveC57BL-6 mice through the jugular vein at a dose of 1.0 × 10¹⁰ particlesper gram/body weight ⁴⁸. Particle count was measured by NanosightTracking analysis and samples were diluted accordingly. Prior to surgeryanimals were anesthetized with ketamine and xylene. A 1-2 cm incisionwas made between the jaw and the clavicle. The jugular vein was elevatedand tied, followed by catheter placement. Serum-derived EV weretransferred and lung and brain tissues were collected 24 hours afterinjection for analysis (n=5).

Enoxaparin and Anti-ASC Treatment

Serum-derived EV from TBI mice were injected into naïve C57-BL6 micethrough a jugular vein injection. One hour later, Enoxaparin (3 mg/kg)(n=4) and Anti-ASC (5 mg/kg) (n=4) were administered to recipientanimals. The following groups were used: 1) the naïve group received notreatment, 2) the sham saline group was used as a negative control andunderwent jugular vein injection of only saline, 3) the untreated groupreceived EV from TBI mice without any treatment and was used as apositive control, 4) the ENOX group received EV from TBI mice andEnoxaparin, and 5) the Anti-ASC group received EV from TBI mice andAnti-ASC. The order of treatment was randomized. Lung and brain tissueswere collected 24 hours after injection for analysis.It should be notedthat the anti-ASC antibody used in the treatment experiments was ahumanized monoclonal antibody against ASC and recognizes murine, humanand swine ASC.

Histology and Lung Injury Scoring

Lung tissue sections were stained by a standard hematoxylin and eosinmethod for histology, morphometry and ALI scoring. Lung sections werescored by a blinded pathologist using the Lung Injury Scoring Systemfrom the American Thoracic Society Workshop Report (17). Twenty randomhigh power fields were chosen for scoring. Criteria for ALI scoring wasbased on number of neutrophils in the alveolar space, interstitialspace, hyaline membranes, proteinaceous debris filling the airspaces andalveolar septal thickening. Based on these criteria a score between 0(no injury) and 1 (severe injury) was given.

Statistical Analysis

Data were analyzed using a student’s T-test for two groups and a one-wayANOVA followed by Tukey’s multiple comparison tests, (GraphPad Prismversion 7.0) for two or more groups. D′Agostino-Pearson test was used totest for normality. Data are expressed as mean +/-SEM. P values ofsignificance used were * p<0.05.

Results Severe TBI Increases AIM2 Inflammasome Proteins and HMGB1Expression in the Brain Of Mice

Excessive levels of the proinflammatory cytokine IL-1β and IL-18, andinflammasome proteins are associated with secondary damage afterfluid-percussion brain injury (18). To determine whether severe CCIinduced processing of proinflammatory cytokines and alterations inlevels of inflammasome proteins, cortical lysates were analyzed, howeverthere is limited research on inflammasome activation in severe TBI. Inthis Example, following severe CCI, cortical lysates were examined forthe levels of the caspase-1 (FIGS. 1A, B) (p<0.001), ASC (FIGS. 1A, C)(p=0.003), IL-18 (FIGS. 1A, D) (p=0.0042), AIM2 (FIGS. 1A, F) (p=0.0197)and IL-1β (FIGS. 1A, G) (p=0.0141) at 4 and 24 hrs after injury.. Levelsof caspase-1, ASC, AIM2, and IL-1β peaked at 4 hours after CCI anddecreased by 24 hrs. The time course for maturation of inflammatorycytokines differed slightly but peaked by 24 hours after TBI. Sinceothers have shown a role for the inflammasome DAMP HMGB1activating theAIM2 inflammasome, the levels of these proteins were also determined incortical lysates. As shown in FIGS. 1A, 1E, CCI induced a significantincrease in the levels of HMGB1 (FIGS. 1A, 1E) (p=0.0121) at 4 and 24hrs after injury. These data indicate that following severe CCI in mice,the levels of the AIM2 inflammasome proteins were significantly elevatedin the cortex following injury.

Severe TBI Increases AIM2 Inflammasome Protein and HMGB1 Expression onthe Lungs Of Mice

To determine whether CCI induced inflammasome activation in the lungs,an immunoblot analysis of lung lysates was performed for caspase-1(FIGS. 1H, I) (p=0.0026), ASC (FIGS. 1H, J) (p=0.0427), IL-18 (FIGS. 1H,K) (p=0.0025), IL-1β (FIGS. 1H,N) (p=0.0012) and AIM2 (FIGS. 1H,M)(p<0.001), and NLRP3 (p=0.0047) (Supplemental FIG. 1 ). Increased levelsof caspase-1, ASC, IL-18 and AIM2 were significantly increased at 4 hrsand 24 hrs after injury as compared to the sham control. However thetime course of the increase in protein expression differed slightly fromthat observed in brain in which they peaked at 24 hr after CCI. Since,the HMGB1-RAGE axis plays a role in the mechanism by which TBI induceslung dysfunction (8), lung lysates were analyzed for levels of HMGB1protein expression. FIGS. 1H, 1L (p=0.0158) shows that HMGB1 expressionincreased at 4 and 24 hours after TBI, indicating that the AIM2inflammasome and HMGB1 play a role in the inflammatory response in thelungs post-TBI.

TBI Induces Pyroptosis in the Lungs of Mice

As shown previously, activation of the AIM2 inflatrimasome in corticalneurons leads to pyroptotic cell death (19).To investigate whether TBIresults in pyroptosis in mice lung tissue, the pyroptosome in lungtissue was isolated after TBI. TBI animals, sacrificed at 4 hourspost-injury showed evidence of ASC oligomerization compared to shamanimals (FIG. 4A). ASC dimers, and trimers were seen in TBI animals (50,75 kDA respectively). These results were indicative of pyroptosomeformation, which can be characterized by the supramolecular assembly ofASC oligomers. In addition, gasdermin D (GSDMD), which is cleaved uponactivation of caspase-1 and triggers pyroptosis and the release of IL-1β(20), was significantly increased in the lungs of TBI animals comparedto sham (FIGS. 4B and 4C) (p=0.0001). These findings indicated thatpyroptosis contributes to cell death in lung tissue after TBI.

TBI Increases Immunoreactivity of Inflammasome Proteins in Type IIAlveolar Epithelial Cells

TBI may lead to capillary leak, resulting in increased vascularpermeability and damage to specialized alveolar epithelial cells, calledtype II pneumocytes (5). To examine the cellular effects of TBI oninflammasome expression in the lungs after injury, immunohistochemicalanalysis was performed in lung sections of sham, 4 hour, and 24 hourinjured animals. Type II alveolar epithelial cells are known to be themain type of lung cells injured in ALl (17). Lung sections were stainedwith antibodies against AIM2, caspase-1, and ASC (green) and co-stainedwith Pro-surfactant protein C (Pro-SPC, red), a marker of type IIepithelial cells, and DAPI nuclear staining (blue). As shown in FIGS.2A-2C, active caspase-1 (FIG. 2A), ASC (FIG. 2B), as well as AIM2 (FIG.2C) are present in SPC-positive cells (arrow). Immunoreactivity of theseinflammasome proteins increased after TBI. These findings indicate thatinflammasome proteins are expressed in type II alveolar epithelial cellsand that TBI results in increased immunoreactivity in these cells.

TBI Increases Nuclear and Cytoplasmic HMGB1 Expression

In order to determine the cellular distribution of HMGB1 in lung cellsafter TBI, nuclear and cytoplasmic fractions from lung homogenates wereisolated (FIGS. 3A, 3C) (p=0.0337). Immunoblotting indicated that bothfractions had significant increases in HMGB1 expression at 4 hrspost-TBI (FIGS. 3B, 3D) (p=0.0345). Immunohistochemical analysis ofHMGB1 was also performed in order to determine the changes inimmunoreactivity in lung sections after TBI. Sections were co-stainedfor HMGB1 (green) and SPC (red) and DAPI nuclear staining (blue).Immunoreactivity of HMGB1 was increased at 4 hrs and 24 hrs whencompared to sham. Weak immunoreactivity of HMGB1 was observed inSPC-positive cells (arrow) (FIG. 3E); therefore, suggesting that HMGB1changes in the injured lung tissue may be cytoplasmic.

TBI Induces Changes in Lung Morphology and Induces ALI

ALI can be characterized by inflammatory processes, which lead toalveolar and interstitial edema as well as infiltration of inflammatorycells into the alveolar space (23). Histopathological analysis of lungtissue (FIG. 5A) indicate that severe TBI causes substantial changes inthe lung architecture and morphology at 4 and 24 hours after injury.Sham animals showed a normal alveolar morphology, whereas injuredanimals showed acute changes in alveolar edema but decreased slightly by24 hours after injury (long arrows). In addition, there was evidence ofneutrophil infiltration (arrow heads) and changes in morphology ofalveolar capillary membranes (*) at both time points. Injured animalsshowed signs of interstitial edema, which was more pronounced at 4 hourspost-injury, but was still evident at 24 hours post injury (shortarrows). Lastly, injured animals also showed evidence of thickening ofthe interstitial area and the alveolar septum (pound, #).

To confirm that severe injury induces ALI, histological sections wereanalyzed using the ALI scoring system defined by the American ThoracicSociety (17). This system is based on evidence of neutrophilinfiltration into the alveolar and interstitial spaces, hyaline membraneformation, proteinaceous debris filling the airspaces, and alveolarseptal thickening.(17). These characteristics were significantlyelevated in injured animals and ALI scores were higher overall in TBIanimals compared to sham (FIG. 5B) (p=0.0017).

Enoxaparin and Anti-ASC Antibody Treatment Significantly ReducesInflammasome Expression and ALI After Adoptive Transfer of EV from TBIMice

In order to provide evidence that EV and their cargo that can bereleased into the circulation after TBI may induce inflammasomeactivation in the lung, a classic adoptive transfer experiment wasperformed using serum-derived EV from severe CCI mice. EV preparationswere validated using Western Blot for EV marker CD81 (FIG. 6 ). Controlsreceived EV isolated from sham or naive animals. As shown in FIGS.7A-7F, active caspase-1 (FIGS. 7A, 7B), ASC (FIGS. 7A, 7C), IL-18 (FIGS.7A, 7D), AIM2 (FIGS. 7A, 7E) and HMGB1 (FIGS. 7A, 7F) were significantlyelevated in the lungs of animals that received the EV from TBI injuredanimals when compared to the lungs of animals that receive EV fromuninjured or naive mice or naive mice. Furthermore, infiltration ofinflammatory cells (arrows) was apparent in lungs treated with EV fromTBI mice (FIG. 7G). Lastly, ALI score was also significantly higher inanimals that received EV from injured mice (FIG. 7H). These studiesprovided evidence for a neural-respiratory-inflammasome axis in which EVreleased into the circulation after TBI activate the inflammasome inlung target cells contributing to the pathogenesis of ALI.

Next, exosome uptake blockade was attempted by treatment with eitherEnoxaparin or a monoclonal antibody against ASC after adoptive transferof EV from injured to naive mice. Negative control animals receivedsaline and positive control animals received no treatment. As shown inFIGS. 8A-8F, Caspase-1 (FIGS. 8A, 8B), ASC (FIGS. 8A, 8C), IL-1β (FIGS.8A, 8D), AIM2 (FIGS. 8A, 8E), and HMGB1 (FIGS. 8A, 8F) weresignificantly reduced (p=<0001) as compared to untreated (positivecontrol) group after treatment with Enoxaparin or a humanized monoclonalanti-ASC antibody (e.g. IC 100 antibody). In addition, H&E stained lungsections showed significantly less neutrophil infiltration into alveolarand interstitial space, as well as no signs of septal thickening (FIGS.9A-D). ALI scores for animals treated with Enoxaparin and anti-ASCantibody (IC 100) were significantly lower compared to untreated group(FIG. 9E) (p=<0.0001). Thus, EV released into the circulation after TBIplay a role in inflammasome activation in lung cells leading to ALI.

Conclusions

TBI can be associated with higher rates of certain medicalcomplications, especially pulmonary and central nervous systemdysfunction. In this Example, severe TBI was shown to increase HMGB1 andinflammasome expression (e.g., AIM2, caspase-1 and ASC expression) incortical and lung tissue and induce changes in lung morphologyconsistent with ALI (e.g., infiltration of neutrophils into the alveolarand interstitial space, alveolar septal thickening, and alveolar edemaand hemorrhage) and introduces the idea of a Neural RespiratoryInflammatory Axis. Importantly, TBI resulted in pyroptosis in lungtissue (e.g., presence of GSDMD cleavage and increased expression ofinflammasome proteins in Type II alveolar epithelial cells.Additionally, adoptive transfer of EV from TBI mice activated theinflammasome and induced ALI, indicating that brain injury induces therelease of EV containing a cargo of inflammasome proteins that are thencarried to the resulting in ALI. Moreover, it was shown that by bothinhibiting EV uptake (Enoxaparin) and inflammasome activation (anti-ASCantibody (IC 100) treatment), there is a reduction in inflammasomeprotein expression and in the development of ALI.

In summary, this Example showed that AIM2 inflammasome signaling plays acentral role in the pathomechanism of lung injury after TBI anddemonstrates a mechanism of TBI-induced ALI involving EV-mediatedinflammasome signaling. These data provided evidence that EV-mediatedinflammasome signaling can play a central role involving aNeuronal-Respiratory-Inflammatory Axis. Therefore, targeting this axiswith antibodies against inflammasome proteins or drugs that block EVuptake may provide a therapeutic approach in Neurotrauma-induced ALI inall areas of critical care medicine. In light of these results, thedisclosed therapeutic strategies may be useful for the treatment ofinflammatory diseases of the lung in general.

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Example 2: Role of EV Mediated Inflammasome Signaling in ALI FollowingTBI in Human Patients

As a follow up to the experiments on mice in Example 1, the role of EVisolated from human TBI patients on inflammasome signaling in humanpulmonary endothelial cells was examined.

In a first experiment, serum-derived EV were isolated from TBI andcontrol patients using Total Exosome Isolation kit (Thermofisher).Pulmonary Human Microvascular Endothelial Cells (HMVEC-Lonza) werecultured and plated on a 12-well plate. After confluency was reached,isolated EV from TBI and control patients were delivered (1.94 × 108particles/ml) to cells for an incubation period of 4 hours. Afterincubation cells were harvested with 200 ul of lysis buffer and celllysates were used for Western Blot analysis.

In a second experiment, serum-derived EV were isolated from TBI andcontrol patients using Total Exosome Isolation kit (Thermofisher).Pulmonary Human Microvascular Endothelial Cells (HMVEC- Lonza) werecultured and plated on a 96-well plate. After confluency was reached,isolated EV from TBI and control patients were delivered (1.94 × 108particles/ml) to cells for an incubation period of 3 hours and then 1additional hour with caspase-1 FAM FLICA (ImmunohistochemistryTechnologies) with a 1:30 volume to volume ratio. After incubation,media was removed and cells were washed 3 times with apoptosis washbuffer (Immunohistochemistry Technologies). Cells were then co-stainedwith Hoechst for nuclear staining and Propidium Iodide for cell death.Images were taken using an EVOS microscope and then cells were readunder a fluorescent plate reader at an excitation wavelength of 492 nmand an emission wavelength of 520 nm.

Results

As shown in FIGS. 10A-10F, delivery of serum-derived EV from TBIpatients increased inflammasome protein expression in pulmonaryendothelial cells. FIGS. 10A-10E showed that caspase-1, ASC, AIM2, andHMGB 1 were elevated in PMVEC incubated with TBI-EV for 4 hours ascompared to PMVEC incubated with control-EV for 4 hours. Immunoassayresults showed a significant increase in IL-1beta expression using Ellasimple plex assay (FIG. 10F).

As shown in FIGS. 11A-11C, delivery of TBI-EV to pulmonary endothelialcells increased immunoreactivity of caspase-1 and cell death.

Conclusion

These studies provided further evidence for aneural-respiratory-inflammasome axis in which EV released into thecirculation after TBI activate the inflammasome in lung target cellscontributing to the pathogenesis of ALI.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent application, foreign patents, foreign patentapplication and non-patent publications referred to in thisspecification are incorporated herein by reference, in their entirety.Aspects of the embodiments can be modified, if necessary to employconcepts of the various patents, application and publications to provideyet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

What is claimed is:
 1. A method of treating inflammation in lungs of apatient in need thereof, the method comprising: administering to thepatient a composition comprising an agent that inhibits inflammasomesignaling, whereby the inflammation in the lungs of the patient istreated.
 2. The method of claim 1, wherein the inflammation in the lungsis caused by a condition selected from a central nervous system (CNS)injury, a neurodegenerative disease, an autoimmune disease, asthma,chronic obstructive pulmonary disease, cystic fibrosis, interstitiallung disease and acute respiratory distress syndrome.
 3. The method ofclaim 2, wherein the CNS injury is selected from the group consisting oftraumatic brain injury (TBI), stroke and spinal cord injury (SCI). 4.The method of 2, wherein the neurodegenerative disease is selected fromthe group consisting of amyotrophic lateral sclerosis (ALS), multiplesclerosis (MS) and Parkinson’s disease (PD).
 5. The method of any one ofthe above claims, wherein the administration of the composition resultsin inhibition of inflammasome activation in lung cells of the patient.6. The method of any one of claims 1-4, wherein the administration ofthe composition results in a reduction of caspase-1, nucleotide-bindingleucine-rich repeat pyrin domain containing protein 1 (NLRP1),nucleotide-binding leucine-rich repeat pyrin domain containing protein 2(NLRP2), nucleotide-binding leucine-rich repeat pyrin domain containingprotein 3 (NLRP3), NLR family CARD domain-containing protein
 4. (NLRC4),caspase-11, X-linked inhibitor of apoptosis protein (XIAP), pannexin-1,Apoptosis-associated Spec-like protein containing a Caspase ActivatingRecruitment Domain (ASC), interleukin-18 (IL-18), high mobility groupbox 1 (HMGB1) or absent in melanoma 2 (AIM2) levels in lung cells of thepatient as compared to a control, wherein the control is an untreatedpatient.
 7. The method of claim 5 or 6, wherein the lung cells are TypeII alveolar cells.
 8. The method of any one of claims 1-5, wherein theadministration of the composition results in a reduction in acute lunginjury (ALI) as compared to a control, wherein the control is anuntreated patient.
 9. The method of claim 8, wherein the reduction inALI is evidenced by a reduction in neutrophil infiltration into alveolarand/or interstitial space, reduced or absent alveolar septal thickeningor a combination thereof.
 10. The method of any one of the above claims,wherein the agent is an extracellular vesicle (EV) uptake inhibitor, anantibody that binds to an inflammasome component or a combinationthereof.
 11. The method of claim 10, wherein the EV uptake inhibitor isa compound or an antibody, wherein the antibody is selected fromTable
 1. 12. The method of any of claims 10-11, wherein the agent is anEV uptake inhibitor in combination with an antibody that binds to aninflammasome component.
 13. The method of claim 12, wherein the EVuptake inhibitor is a heparin.
 14. The method of claim 13, wherein theheparin is Enoxaparin.
 15. The method of any of claims 10-14, whereinthe antibody that binds to an inflammasome component is an antibody thatspecifically binds to a component of a mammalian AIM2, NLRP1, NLRP2,NLRP3 or NLRC4 inflammasome.
 16. The method of claim 10 or 15, whereinthe inflammasome component is caspase-1, ASC or AIM2.
 17. The method ofclaim 16, wherein the inflammasome component is ASC.
 18. The method ofclaim 17, wherein the antibody binds to an N-terminal PYRIN-PAAD-DAPINdomain (PYD), C-terminal caspase-recruitment domain (CARD) domain or anepitope derived from the PYD or CARD domain of the ASC protein.
 19. Themethod of claim 17, wherein the antibody binds to an amino acid havingat least 85% sequence identity with an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1 and SEQ ID NO:
 2. 20. The method ofany of claims 17-19, wherein the antibody inhibits ASC activity in thelungs of the patient.
 21. The method of any one of the above claims,wherein the composition is formulated with a pharmaceutically acceptablecarrier or diluent.
 22. The method of any one of the above claims,wherein the composition is administered intracerebroventricularly,intraperitoneally, intravenously or by inhalation.
 23. A method oftreating inflammation in lungs of a patient that has been subjected to acentral nervous system (CNS) injury, the method comprising:administering to the patient a composition comprising an agent thatinhibits inflammasome signaling, whereby the inflammation in the lungsof the patient is treated.
 24. The method of claim 23, wherein the CNSinjury is selected from the group consisting of traumatic brain injury(TBI), stroke and spinal cord injury (SCI).
 25. The method of any one ofclaims 23-24, wherein the administration of the composition results ininhibition of inflammasome activation in lung cells of the patient. 26.The method of any one of claims 23-24, wherein the administration of thecomposition results in a reduction of caspase-1, NLRP1, NLRP2, NLRP3,NLRC4, caspase-11, XIAP, pannexin-1, Apoptosis-associated Spec-likeprotein containing a Caspase Activating Recruitment Domain (ASC),interleukin-18 (IL-18), high mobility group box 1 (HMGB1) or absent inmelanoma 2 (AIM2) levels in lung cells of the patient as compared to acontrol, wherein the control is an untreated patient.
 27. The method ofclaim 25 or 26, wherein the lung cells are Type II alveolar cells. 28.The method of any one of claims 23-27, wherein the administration of thecomposition results in a reduction in acute lung injury (ALI) ascompared to a control, wherein the control is an untreated patient. 29.The method of claim 28, wherein the reduction in ALI is evidenced by areduction in neutrophil infiltration into alveolar and/or interstitialspace, reduced or absent alveolar septal thickening or a combinationthereof.
 30. The method of any one of claims 23-29, wherein the agent isan extracellular vesicle (EV) uptake inhibitor, an antibody that bindsto an inflammasome component or a combination thereof.
 31. The method ofclaim 30, wherein the EV uptake inhibitor is a compound or an antibody,wherein the antibody is selected from Table
 1. 32. The method of any ofclaims 30-31, wherein the agent is an EV uptake inhibitor in combinationwith an antibody that binds to an inflammasome component.
 33. The methodof claim 32, wherein the EV uptake inhibitor is a heparin.
 34. Themethod of claim 33, wherein the heparin is Enoxaparin.
 35. The method ofany of claims 30-34, wherein the antibody that binds to an inflammasomecomponent is an antibody that specifically binds to a component of amammalian AIM2, NLRP1, NLRP2, NLRP3 or NLRC4 inflammasome.
 36. Themethod of claim 30 or 35, wherein the inflammasome component iscaspase-1, ASC or AIM2.
 37. The method of claim 36, wherein theinflammasome component is ASC.
 38. The method of claim 37, wherein theantibody binds to the PYD, CARD domain or an epitope derived from thePYD or CARD domain of the ASC protein.
 39. The method of claim 37,wherein the antibody binds to an amino acid having at least 85% sequenceidentity with an amino acid sequence selected from the group consistingof SEQ ID NO: 1 and SEQ ID NO:
 2. 40. The method of any of claims 37-39,wherein the antibody inhibits ASC activity in the lungs of the patient.41. The method of any one of claims 23-40, wherein the composition isformulated with a pharmaceutically acceptable carrier or diluent. 42.The method of any one of claims 23-41, wherein the composition isadministered intracerebroventricularly, intraperitoneally, intravenouslyor by inhalation.